One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as phosphatases. Phosphorylation of proteins occurs predominantly at serine, threonine, or tyrosine residues and protein kinases have therefore been classified by their specificity of phosphorylation site i.e. serine/threonine kinases and tyrosine kinases. Because phosphorylation is such a ubiquitous process within cells and because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
Akt-2 (also known as PKB beta and RAC-PK beta) is a member of the AKT/PKB family of serine/threonine kinases and has been shown to be overexpressed in several types of cancers as well as a mediator of normal cellular functions.
Akt-2, like other members of the AKT/PKB family, is located in the cytosol of unstimulated cells and translocates to the nucleus following stimulation by several ligands including mitogens and survival factors (Meier et al., J. Biol. Chem., 1997, 272, 30491-30497). Other studies have shown that this activation is through PI3 kinase which is wortmannin sensitive (Franke et al., Science, 1997, 275, 665-668). It is through the pleckstrin homology domain (PH) within the protein that Akt-2 binds to the lipid products of PI3 kinase allowing presentation of Akt-2 to its upstream activators by directing its translocation to the membrane. Phosphorylation of Akt-2 is necessary for its activation and the kinase responsible for this activation is PDK1 (Cohen et al., FEBS Lett., 1997, 410, 3-10). Once localized to the membrane, Akt-2 mediates several functions within the cell including the metabolic effects of insulin (Calera et al., J. Biol. Chem., 1998, 273, 7201-7204), induction of differentiation and/or proliferation, protein synthesis and stress responses (Alessi and Cohen, Curr. Opin. Genet. Dev., 1998, 8, 55-62).
Manifestations of altered Akt-2 regulation appear in both injury and disease, the most important role being in the development of cancer. The first description of Akt-2 was in association with human ovarian carcinomas where the expression of the protein was found to be amplified in 15% of the cases (Cheng et al., Proc. Natl. Acad. Sci. U S A, 1992, 89, 9267-9271). It was also found to be overexpressed in 12% of pancreatic cancers (Cheng et al., Proc. Natl. Acad. Sci. U S A, 1996, 93, 3636-3641). In addition, Bellacosa et al. demonstrated Akt-2 overexpression in 12.1% of ovarian carcinomas and 2.8% of breast carcinomas, and that the amplification was especially frequent in undifferentiated tumors (50%) suggesting that Akt-2 may be associated with tumor aggressiveness (Bellacosa et al., Int. J. Cancer, 1995, 64, 280-285).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Akt-2. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting Akt-2 function.
To date, strategies aimed at inhibiting Akt-2 function have involved the use of antibodies and transfection of antisense constructs of Akt-2 in tumor cell lines. Cheng et al. demonstrated in studies of tumor transplants using nude mice, that invasiveness and tumorigenicity were reduced in tumors transfected with antisense constructs expressing a 1.2 kb fragment of Akt-2 cDNA in the antisense orientation as compared to those cell lines not containing the Akt-2 antisense construct (Cheng et al., Proc. Natl. Acad. Sci. U S A, 1996, 93, 3636-3641).
Antisense oligonucleotides, therefore, provide a promising new pharmaceutical tool for the effective modification of the expression of specific genes including Akt-2.